23Bovine colostrum (COL) has been advocated as a nutritional countermeasure to 24 exercise-induced immune dysfunction and increased risk of upper respiratory illness 25 (URI) in athletic populations, however, the mechanisms remain unclear. During winter 26 months, under double-blind procedures, 53 males (mean training load ± SD, 50.5 ± 27 28.9 MET-hweek -1 ) were randomized to daily supplementation of 20 g of COL (N = 28 25) or an isoenergetic/isomacronutrient placebo (PLA) (N = 28) for 12 weeks. Venous 29 blood was collected at baseline and at 12 weeks and unstimulated saliva samples at 30 4-week intervals. There was a significantly lower proportion of URI days and number 31 of URI episodes with COL compared to PLA over the 12 weeks (p < 0.05). There was 32

Bacterial growth and proliferation can be restricted by limiting the availability of metal ions in their environment. Humans sequester iron, manganese and zinc to help prevent infection by pathogens, a system termed nutritional immunity. Commercially-used chelants have high binding affinities with a variety of metal ions, which may lead to antibacterial properties that mimic these innate immune processes. However, the modes of action of many of these chelating agents in bacterial growth inhibition and their selectivity in metal deprivation in cellulo remain ill-defined. We address this shortcoming by examining the effect of eleven chelators on Escherichia coli growth and their impact on the cellular concentration of five metals. Four distinct effects were uncovered: i) no apparent alteration in metal composition, ii) depletion of manganese alongside reductions in iron and zinc levels, iii) reduced zinc levels with a modest reduction in manganese, and iv) reduced iron levels coupled with elevated manganese. These effects do not correlate with the absolute known chelant metal ion affinities in solution, however, for at least five chelators for which key data are available, they can be explained by differences in the relative affinity of chelants for each metal ion. The results reveal significant insights into the mechanism of growth inhibition by chelants, highlighting their potential as antibacterials and as tools to probe how bacteria tolerate selective metal deprivation. IMPORTANCE Chelating agents are widely used in industry and consumer goods to control metal availability, with bacterial growth restriction as a secondary benefit for preservation. However, the antibacterial mechanism of action of chelants is largely unknown, particularly with respect to the impact on cellular metal concentrations. The work presented here uncovers distinct metal starvation effects imposed by different chelants on the model Gram-negative bacterium Escherichia coli . The chelators were studied both individually and in pairs with the majority producing synergistic effects in combinations that maximise antibacterial hostility. The judicious selection of chelants based on contrasting cellular effects should enable reductions in the quantities of chelant required in numerous commercial products and presents opportunities to replace problematic chemistries with biodegradable alternatives.

EDTA is widely used as an inhibitor of bacterial growth, affecting the uptake and control of metal ions by microorganisms. We describe the synthesis and characterisation of two symmetrical bis-amide derivatives of EDTA, featuring glycyl or pyridyl substituents: AmGly and AmPy . Metal ion affinities (logK) have been evaluated for a range of metals (Mg , Ca , Fe , Mn , Zn ), revealing less avid binding compared to EDTA. The solid-state structures of AmGly and of its Mg complex have been determined crystallographically. The latter shows an unusual 7-coordinate, capped octahedral Mg centre. The antibacterial activities of the two ligands and of EDTA have been evaluated against a range of health-relevant bacterial species, three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and a Gram positive (Staphylococcus aureus). The AmPy ligand is the only one that displays a significant inhibitory effect against K. pneumoniae, but is less effective against the other organisms. AmGly exhibits a more powerful inhibitory effect against E. coli at lower concentrations than EDTA (<3 mm) or AmPy , but loses its efficacy at higher concentrations. The growth inhibition of EDTA and AmGly on mutant E. coli strains with defects in outer-membrane lipopolysaccharide (LPS) structures has been assessed to provide insight into the unexpected behaviour. Taken together, the results contradict the assumption of a simple link between metal ion affinity and antimicrobial efficacy.

Chelating ligands such as EDTA are widely added to consumer products for preservation enhancement. They inhibit the growth of bacteria and other microorganisms by interfering with the uptake and usage of essential metal ions, though the precise mechanisms by which they do so aren't well understood. Two di‐amide derivatives of EDTA are prepared and characterised in this study that feature additional ligating groups in the pendent amide arms. Under some conditions, these ligands reveal superior bacteriostatic activity to EDTA, despite being less avid binders of common bio‐relevant metal ions. Minimum inhibitory concentrations (MIC), at which they exert bacteriostatic effects, do not necessarily correlate with log K values for metal ion binding, challenging the conventional wisdom. Growth inhibition on mutant E. coli strains with defective outer‐membrane lipopolysaccharide structures is assessed to provide insight into the unexpected behaviour. For more details, see the Full Paper by J. A. Gareth Williams et al. on page 7137 ff.